Previously, we have examined the signalling mechanisms for the release of inflammatory mediators in antigen stimulated RBL-2H3 mast cells and shown that production of inflammatory cytokines and arachidonic acid is regulated primarily by the p42 MAP kinase, ERK2,whereas secretion of the newly formed cytokines via Golgi and preformed secretory granules is regulated primarily by protein kinase C and calcium. We now find that antigen-stimulation increases expression of COX-2 but not of the other enzymes involved in production of inflammatory eicosanoids from arachidonic acid. This induction correlates with the activation of the p38 MAP kinase and is blocked by inhibitors of the p38 MAP kinases and by low concentrations of the anti-inflammatory steroid, dexamethasone. Dexamthasone suppresses activation of p38 MAP kinase as well as other MAP kinase cascades at the same low concentrations. We had previously shown that dexamethasone blocks activation of the ERK MAP kinase cascade by inhibiting the interaction of Ras with the enzyme that initiates this cascade, namely Raf-1, without affecting upstream events. Indeed, dexamethasone appears to suppress activation of all counterparts of Raf-1 in the various MAP kinase cascades by causing their disocciation from heat shock protein 90(Hsp90). These findngs are novel and we have used Raf-1 to examine this phenomenon in detail. Dexamethasone appears to target Hsp90 as other chaperone proteins such as such Hsp70, 14-3-3, cdc37, p50 and FKBP65 remain attached to Raf-1. Raf-1 bereft of Hsp90 fails to associate with membrane-bound Ras even though Ras is activated and associates with other proteins such as PI 3-kinase in activated RBL-2H3 cells. We believe these results reveal an unsuspected action of dexamethasone and a critical role for Hsp90 in MAP kinase signalling. To determine the relevance of the above findings in normal mast cells, we are currently characterizing growth of mast cells derived from human progentitor CD24+ cells grown in the presence of IL-3, IL-6 and stem cell factor (SCF). Proliferation of these cells is associated with a sharp but transient increase in telomerase activity (dependent on SCF and PI-3kinase)which rapidly declines once cells differentiate. The mature mast cells secrete histamine and cytokines in response to antigen stimulation and exhibit normal signalling mechanisms as indicated by tyrosine phosphorylation of proteins. The effects dexamethasone on the Ras/Raf/ERK cascade is now under study (Studies performed in collaboration with Drs. Gilfillan and Metcalfe, NIAID). - mast cell cultures, cytokines, aracidonic acid, MAP kinases, heat shock proteins, dexamethasone